Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy Fares Bassila,b, Pierre-Olivier Fernaguta,b, Erwan Bezarda,b, Alain Pruvostc, Thierry Leste-Lasserred, Quyen Q. Hoange, Dagmar Ringef, Gregory A. Petskof,g,1, and Wassilios G. Meissnera,b,h,1 aUniversité de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; bCNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; cCommissariat à l’énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et de Technologies de Saclay (iBiTec-S), Service de Pharmacologie et d’Immunoanalyse (SPI), Université Paris Saclay, F-91191 Gif sur Yvette, France; dINSERM, Neurocentre Magendie, U1215, Physiopathologie de la Plasticité Neuronale, F-33000 Bordeaux, France; eDepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202; fRosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02465; gAppel Alzheimer’s Disease Research Institute, Weill Cornell Medical College, New York, NY 10021; and hCentre de Référence Maladie Rare Atrophie Multisystématisée, Service de Neurologie, Hôpital Pellegrin, Centre Hospitalier Universitaire de Bordeaux, F-33076 Bordeaux, France Contributed by Gregory A. Petsko, June 13, 2016 (sent for review June 5, 2015; reviewed by Peter T. Lansbury and Sidney Strickland) Multiple system atrophy (MSA) is a sporadic orphan neurodegen- MSA (and other synucleinopathies) (15) by decreasing α-syn olig- erative disorder. No treatment is currently available to slow down omerization and aggregation. Interestingly, the inflammatory pro- the aggressive neurodegenerative process, and patients die within tease caspase-1 cleaves α-syn at Asp121, promoting its aggregation a few years after disease onset. The cytopathological hallmark of into amyloid fibrils similar to those previously found both in vitro MSA is the accumulation of alpha-synuclein (α-syn) aggregates in and in vivo (16). In turn, the caspase-1 inhibitor prodrug VX-765 affected oligodendrocytes. Several studies point to α-syn oligomer- decreases α-syntruncationandaggregationinvitroandrescues ization and aggregation as a mediator of neurotoxicity in synuclei- cells from α-syn–induced toxicity (16). Therefore, VX-765 could nopathies including MSA. C-terminal truncation by the inflammatory exert neuroprotective effects on MSA pathogenesis by reducing protease caspase-1 has recently been implicated in the mechanisms α-syn cleavage, hence limiting its toxicity and its ability to form ag- that promote aggregation of α-syn in vitro and in neuronal cell models gregates. VX-765 is an orally active, well-tolerated, brain-penetrant MEDICAL SCIENCES of α-syn toxicity. We present here an in vivo proof of concept of the prodrug that is hydrolyzed by esterases in vivo to produce a potent ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn and selective caspase-1 inhibitor (17, 18), an activity supported by – pathology and to mediate neuroprotection in proteolipid protein the in vivo demonstration of reduced cleavage of pro Interleukin- β β β α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and 1 (IL-1 ) to its activated form IL-1 , a proinflammatory process – age-matched wild-type mice were treated for a period of 11 wk with under control of caspase-1 (18 20). VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice Here we show that the brain-penetrant VX-765 mitigates compared with placebo controls. More importantly, VX-765 was able progressive synucleinopathy and neurodegeneration in a trans- to limit the progressive toxicity of α-syn aggregation by reducing its genic mouse model of MSA. load in the striatum of PLP-SYN mice. Not only did VX-765 reduce Results truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserv- VX-765 Prevents Motor Impairments in Transgenic MSA Proteolipid α α ing tyrosine hydroxylase-positive neurons in the substantia nigra Protein -Syn Mice. Transgenic MSA proteolipid protein -syn of PLP-SYN mice. In conclusion, our results suggest that VX-765, a (PLP-SYN) mice display progressive motor impairment with drug that was well tolerated in a 6 wk-long phase II trial in pa- tients with epilepsy, is a promising candidate to achieve disease Significance modification in synucleinopathies by limiting α-syn accumulation. Multiple system atrophy (MSA) is a fatal neurodegenerative disorder alpha-synuclein | multiple system atrophy | caspase-1 | truncation associated with the accumulation of alpha-synuclein (α-syn) aggre- gates in oligodendrocytes. There is currently no treatment to slow ultiple system atrophy (MSA) is a sporadic adult-onset down the aggressive neurodegenerative process. C-terminal trun- Morphan neurodegenerative disorder clinically characterized cation of α-syn promotes the formation of oligomers and ag- by a combination of parkinsonism, cerebellar impairment, and au- gregates that, in turn, mediate neurotoxicity in synucleinopathies tonomic dysfunction (1). The cytopathological hallmark of MSA is including MSA. We present here an in vivo proof of concept of the α the accumulation of alpha-synuclein (α-syn) aggregates in oli- ability of the caspase-1 inhibitor VX-765 to mitigate -syn pathology and provide neuroprotection in a transgenic mouse model of MSA godendrocytes, forming glial cytoplasmic inclusions (GCIs) (2, 3). α The 14-kDA protein α-syn can exist in vitro as an unfolded through reduction of -syn C-terminal truncation. These findings monomer; although other oligomeric species have been reported suggest that VX-765, a well-tolerated drug in a 6 wk-long phase II (4). Full-length α-syn undergoes several posttranslational modi- trial in patients with epilepsy, is a promising candidate to achieve α fications such as phosphorylation, tyrosine nitration, and trun- disease modification in MSA by limiting -syn accumulation. cation, any of which could promote the formation of toxic α-syn – α Author contributions: P.-O.F., E.B., and W.G.M. designed research; F.B., A.P., T.L.-L., and aggregates (5 7). Although the precise toxic species of -syn have W.G.M. performed research; Q.Q.H. and G.A.P. contributed new reagents/analytic tools; not been firmly established, several studies point to α-syn oligo- F.B., P.-O.F., E.B., A.P., T.L.-L., and W.G.M. analyzed data; and F.B., P.-O.F., E.B., Q.Q.H., D.R., merization and aggregation as a mediator of neurotoxicity in G.A.P., and W.G.M. wrote the paper. synucleinopathies (6, 8–10). Hence, decreasing aggregation Reviewers: P.T.L., Harvard Medical School; and S.S., The Rockefeller University. might be an effective approach to disease modification. Among The authors declare no conflict of interest. α the mechanisms that promote aggregation of -syn, C-terminal 1To whom correspondence may be addressed. Email: [email protected] truncation has been identified as an enhancer/promoter of or [email protected]. α – -syn oligomerization and fibrillization (11 14). Accordingly, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inhibiting α-syn truncation could alter the disease course in 1073/pnas.1609291113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1609291113 PNAS | August 23, 2016 | vol. 113 | no. 34 | 9593–9598 Downloaded by guest on October 2, 2021 Fig. 1. VX-765 treatment reversed α-syn–induced pathology in PLP-SYN mice by decreasing α-syn load in the striatum and rescuing motor performance. (A) Placebo-treated PLP-SYN mice produced more errors per step compared with VX-765–treated PLP-SYN mice in the challenging beam test. (B–F) Repre- sentative immunoblot levels of oligomeric (D), monomeric (E), and truncated (F) α-syn in placebo- (B) and VX-765– (C) treated PLP-SYN mice. (G) Analysis of human α-syn mRNA levels in VX-765–treated PLP-SYN mice compared with placebo-treated mice. (H–M) Immunohistochemical analysis of α-syn load (H–J)and insolubility (K–M) in the striatum of placebo- (I and K) and VX-765– (J and L) treated PLP-SYN mice. In all panels, n = 8 per experimental group—except for mRNA α-syn (G), n = 5 for placebo-treated and n = 6 for VX-765–treated PLP-SYN mice. Error bars indicate SE. *P < 0.05, **P < 0.01, ***P < 0.001. FL, full length; Hmw, high molecular weight; PK, proteinase-K; Trc, truncated. aging, as shown with an increased number of errors on the tra- between groups, thus indicating no effect of VX-765 treat- versing beam task (21). Motor performance of wild-type (WT) ment on the transcription of human α-syn. mice was not affected by VX-765 treatment (100 mg·kg·dover We then assessed the density of α-syn inclusions in PLP-SYN 11 wk; P = 0.99), whereas VX-765-treated PLP-SYN mice showed mice and whether VX-765 affects α-syn aggregate solubility using significant improvement in the traversing beam task compared with immunohistochemistry on adjacent sections, with or without placebo-treated PLP-SYN mice (P = 0.01) (Fig. 1A). proteinase-K (PK) pretreatment, in the striatum (Fig. 1 H–M) and in the cortex (Fig. 2 H–M). VX-765–treated PLP-SYN mice VX-765 Decreases α-Syn Burden in the Striatum of PLP-SYN Mice. showed a significant decrease in the density of α-syn–immuno- GCIs are the cytopathological hallmark of MSA (3). PLP-SYN positive GCIs in the striatum (–40%, P < 0.001) (Fig. 1 H–J)but mice overexpress α-syn under the PLP promoter, leading to the not in the cortex (P = 0.1) (Fig. 2 H–J) compared with placebo formation of GCIs (21, 22). To investigate whether reducing PLP-SYN mice. The amount of PK-resistant α-syn aggregates was C-terminal truncation affects α-syn load in PLP-SYN mice, we also significantly lowered in the striatum of VX-765–treated PLP- first measured the quantity of α-syn in the striatum (Fig.